WO2015033616A1 - Dispositif d'entraînement de récipients de culture, et système de culture - Google Patents

Dispositif d'entraînement de récipients de culture, et système de culture Download PDF

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Publication number
WO2015033616A1
WO2015033616A1 PCT/JP2014/062353 JP2014062353W WO2015033616A1 WO 2015033616 A1 WO2015033616 A1 WO 2015033616A1 JP 2014062353 W JP2014062353 W JP 2014062353W WO 2015033616 A1 WO2015033616 A1 WO 2015033616A1
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WIPO (PCT)
Prior art keywords
culture
culture vessel
container
vessel
driving device
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PCT/JP2014/062353
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English (en)
Japanese (ja)
Inventor
正和 福島
伸一郎 長澤
浩樹 福冨
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旭化成株式会社
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Application filed by 旭化成株式会社 filed Critical 旭化成株式会社
Priority to JP2015535339A priority Critical patent/JPWO2015033616A1/ja
Publication of WO2015033616A1 publication Critical patent/WO2015033616A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/50—Means for positioning or orientating the apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/02—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by impregnation, e.g. using swabs or loops
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/08—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by vibration
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/02—Separating microorganisms from the culture medium; Concentration of biomass

Definitions

  • the present disclosure relates to a culture container driving device and a culture system.
  • Cell culturing procedures include detaching cells from the inner surface of the culture vessel, such as a step of removing unnecessary cells by detaching from the inner surface of the culture vessel, or a step of separating and recovering the cultured cells from the inner surface of the culture vessel.
  • Process Conventionally, the operation of peeling cells from the inner surface of the culture container is performed by, for example, applying a liquid to the inner surface of the culture container using a pipette (hereinafter referred to as “pipetting”). Since pipetting is performed manually, when trying to cultivate a large number of cells simultaneously using many culture vessels, excessive labor of the operator is required. In addition, since stable pipetting requires skill of workers, it is difficult to secure the number of workers commensurate with labor, and pipetting requires an excessive amount of time. On the other hand, for example, Patent Document 1 discloses an apparatus for automating pipetting.
  • Patent Document 2 discloses a method of peeling cells from the inner surface of a culture container by applying ultrasonic vibration to the culture container.
  • Patent Document 3 discloses a method of peeling cells from the inner surface of a culture container by partially applying a pulsed impact force to the culture container.
  • the present disclosure aims to provide a device capable of automating the work of peeling the deposits on the inner surface of the culture vessel with a simple configuration and improving the cell recovery rate, and a culture system using the device.
  • the culture vessel driving device is the same as the culture vessel holding base having a support surface that supports the culture vessel and a holding unit that holds the culture vessel on the support surface, and the entire culture vessel in the direction orthogonal to the support surface. And a drive unit that vibrates the culture vessel holding table at a frequency of 20 Hz to 2 kHz so as to move simultaneously at the same displacement amount.
  • the culture vessel can be vibrated with a simple configuration in which the culture vessel holding base is vibrated by the drive unit.
  • the culture vessel is vibrated so that the entire culture vessel moves to the same side and with the same amount of displacement at the same time in the direction perpendicular to the support surface.
  • the deposit on the inner surface of the culture vessel is efficiently peeled off. Therefore, the operation of removing the deposits on the inner surface of the culture vessel can be automated with a simple configuration.
  • the reason why the attached matter on the inner surface of the culture vessel is efficiently peeled by the vibration is estimated as follows. That is, by the vibration, a Faraday wave is generated on the liquid level of the culture solution in the culture vessel. In the state where the Faraday wave is generated, a large number of swirling flows are formed in the culture solution along the support surface. Each swirl flow swirls along a plane intersecting the support surface to generate a flow along the inner surface of the culture vessel. By this flow, the deposits on the inner surface of the culture vessel are efficiently peeled off.
  • the drive unit vibrates the culture vessel holding table at a frequency of 20 Hz to 2 kHz.
  • the frequency is lowered, it is difficult to reduce unnecessary vibration transmitted from the drive unit to the surroundings. For this reason, the vibration isolating element for reducing vibration transmission tends to become more complex as the frequency decreases.
  • the energy consumption of the drive unit increases. For this reason, there is a tendency that the power source of the drive unit becomes larger as the frequency becomes higher. If the frequency is 20 Hz to 2 kHz, the deposits on the inner surface of the culture vessel can be peeled off while suppressing the complexity of the vibration isolation element or the increase in the size of the power source of the drive unit.
  • the cell recovery rate is improved as compared with the case where an apparatus for ultrasonically vibrating the culture container is used.
  • the reason for this is estimated as follows. That is, the frequency at which the culture vessel vibrates is much smaller than the ultrasonic frequency. As a result, the energy applied to the cells is small compared to the ultrasonic vibration of the culture vessel, and there are few cells that are crushed by the vibration.
  • the culture container driving device when used, the cell recovery rate is improved as compared with the case where a device that partially applies an impact force to the culture container is used.
  • the reason for this is estimated as follows. That is, when an impact force is partially applied to the culture vessel, the movement of the liquid is large in the portion where the impact is applied, but the movement of the liquid is attenuated in the surrounding area, so that cell detachment tends to be uneven. .
  • the culture container driving device according to the present disclosure, the Faraday wave is generated, so that the deposits on the inner surface of the culture container are efficiently peeled off.
  • the drive unit may vibrate the culture vessel holding table at a frequency of 20 to 100 Hz. In this case, the enlargement of the power source of the drive unit can be further suppressed.
  • the drive unit may vibrate the culture vessel holder so that the amplitude in the direction orthogonal to the support surface is greater than 0 mm and less than 5 mm.
  • the amplitude is increased, the deposits are easily peeled off, while the energy consumption of the drive unit is increased. For this reason, the power source of the drive unit tends to increase in size as the amplitude increases. If the amplitude is more than 0 mm and not more than 5 mm, the deposits on the inner surface of the culture vessel can be peeled off while suppressing an increase in the size of the power source of the drive unit.
  • the culture vessel holding table may have a plurality of shelves that each constitute a support surface. In this case, it is possible to arrange a plurality of culture containers in a direction perpendicular to the support surface, and to vibrate these culture containers at the same time. Thereby, the deposit
  • the culture vessel holder may have a larger support surface than the lower surface of the culture vessel. In this case, it is easy to move the entire culture vessel to the same side at the same time with the same amount of displacement.
  • the drive unit may vibrate the culture vessel holding table under conditions that allow the deposits on the inner surface of the culture vessel to be peeled off without opening the culture vessel.
  • the deposits on the inner surface of the culture container can be peeled off without performing the operation of opening the culture container.
  • the culture vessel holding table holds a culture vessel containing monocytes and lymphocytes, and the drive unit vibrates the culture vessel holding table under the condition that more lymphocytes than monocytes can be detached from the inner surface of the culture vessel. May be.
  • monocytes can be cultured after sufficiently increasing the ratio of monocytes in the culture container by separating and removing lymphocytes adhering to the inner surface of the culture container. Thereby, monocyte differentiation can be efficiently cultured.
  • the drive unit may further vibrate the culture vessel holding table under the condition that the differentiated monocytes can be separated from the inner surface of the culture vessel.
  • the monocyte differentiation adhering to the inner surface of the culture vessel can be efficiently detached and recovered.
  • a culture system includes the culture container driving device, the culture container, an incubator that accommodates the culture container and promotes culture in the culture container, a scraper that scrapes the inner surface of the culture container, and a scraper that drives the scraper.
  • the drive device the vibration peeling controller that controls the culture vessel drive device to vibrate the culture vessel holding base under conditions that allow the deposit on the inner surface of the culture vessel to be peeled off, and the scraper scrape the deposit on the inner surface of the culture vessel.
  • a scraping control unit that controls the scraper driving device so as to be removed.
  • the culture container driving device is controlled by the vibration separation controller, so that the deposits on the inner surface of the culture container can be separated and efficiently recovered. Furthermore, by controlling the scraper driving device by the scraping control unit, the deposits remaining on the inner surface of the culture vessel can be scraped off and collected. Therefore, more deposits can be collected efficiently.
  • the vibration peeling control unit may control the culture container driving device so as to vibrate the culture container holding base under the condition that the deposit on the inner surface of the culture container can be peeled without opening the culture container.
  • the deposits on the inner surface of the culture container can be peeled off without performing the operation of opening the culture container.
  • An infusion device that supplies liquid to the culture container or discharges the liquid from the culture container without opening the culture container held on the culture container holder may be further provided.
  • a step of supplying a liquid such as a culture solution into the culture vessel, a step of peeling off deposits on the inner surface of the culture vessel after the culture, and collecting the peeled deposits together with the liquid A process can be performed. For this reason, culture
  • the scraper may be accommodated in the culture vessel, and the scraper driving device may drive the scraper by magnetic force from the outside of the culture vessel. In this case, it is possible to further perform a step of scraping and collecting deposits on the inner surface of the culture vessel without opening the culture vessel. For this reason, collection
  • the culture vessel contains monocytes and lymphocytes, and the vibration detachment control unit vibrates the culture vessel holder under conditions that allow more lymphocytes to detach from the inner surface of the culture vessel before culturing in the incubator.
  • the culture vessel drive device may be controlled.
  • culturing of monocytes can be promoted in an incubator after sufficiently increasing the ratio of monocytes in the culture container by separating and removing lymphocytes adhering to the inner surface of the culture container. Thereby, monocyte differentiation can be efficiently cultured.
  • the vibration detachment control unit may further control the culture container driving device so as to vibrate the culture container holder under conditions where the monocyte differentiation can be separated from the inner surface of the culture container after culturing in the incubator.
  • the monocyte differentiation adhering to the inner surface of the culture vessel can be efficiently detached and recovered.
  • the incubator and the culture vessel driving device may be provided independently of each other.
  • the culture in the incubator and the detachment of the deposits in the culture container driving device can proceed independently.
  • the culture and stripping cycle is adjusted according to the difference between the time required for culture (hereinafter referred to as “culture time”) and the time required for stripping (hereinafter referred to as “stripping time”).
  • culture time the time required for culture
  • stripping time the time required for stripping
  • the culture time is longer than the stripping time
  • the number of culture vessels placed in the incubator is increased compared to the number of culture vessels placed in the culture vessel drive device, and the stripping cycle is set to the culture cycle. By shortening the length, the culture system can be operated with high efficiency.
  • the work of peeling the deposits on the inner surface of the culture container can be automated with a simple configuration and the cell recovery rate can be improved.
  • FIG. 3 is a sectional view taken along line III-III in FIG. 2.
  • FIG. 4 is a sectional view taken along line IV-IV in FIG. 1.
  • FIG. 5 is a sectional view taken along line VV in FIG. 4.
  • FIG. 7 is a sectional view taken along line VII-VII in FIG. 6. It is a schematic diagram which shows schematic structure of a culture system.
  • the culture vessel drive device 1 includes a culture vessel holding base 2, a drive unit 3, a monitoring unit 4, and a control unit 5.
  • the culture vessel drive device 1 is a device that vibrates a culture vessel 11 containing a liquid and cells, and peels the cells from the inner surface of the culture vessel 11.
  • the culture container driving device 1 is used for selectively separating lymphocytes from the inner surface of the culture container 11 containing monocytes and lymphocytes. Further, it is used to peel dendritic cells (monocyte differentiation) formed by monocyte culture from the inner surface of the culture vessel 11.
  • the culture vessel holder 2 holds the culture vessel 11.
  • the culture vessel 11 has a petri dish shape, and its upper part is closed with a film.
  • a liquid feeding port 11 a for inflowing or outflowing of a liquid such as a culture solution is provided.
  • the culture vessel holder 2 has a four-stage shelf 6 and a bottom plate 7.
  • a columnar spacer 20 is interposed between the shelves 6.
  • the shelf 6 and the spacer 20 may be fixed by bolt fastening or the like, or may be integrated by welding or the like.
  • the bottom plate 7 is disposed below the lowermost shelf 6.
  • a columnar spacer 21 is interposed between the shelf 6 and the bottom plate 7.
  • the shelf 6, the bottom plate 7, and the spacer 21 may be fixed by bolt fastening or the like, or may be integrated by welding or the like.
  • Each of the four shelf sections 6 supports the culture vessel 11.
  • the upper surface M1 of the shelf 6 is larger than the lower surface of the culture vessel 11.
  • Each shelf 6 is provided with a suction portion R1.
  • the adsorption part R1 adsorbs the culture vessel 11 to the upper surface M1 side of the shelf part 6 by negative pressure. That is, the culture vessel holder 2 has a support surface M1 that supports the culture vessel 11 and an adsorption portion R1 that adsorbs the culture vessel 11 to the support surface M1 side by negative pressure.
  • the support surface M1 and the adsorption portion R1 are It is provided for each shelf 6.
  • the adsorption portion R1 functions as a holding portion that holds the culture vessel 11 on the support surface M1.
  • the adsorption part R1 is connected to the vacuum pump 12 via the exhaust pipe L1.
  • the four exhaust pipes L1 respectively extending from the four-stage shelves 6 merge into one exhaust pipe L2 and are connected to the vacuum pump 12.
  • the vacuum pump 12 is, for example, an electric pump, and functions as a negative pressure generation source.
  • Each exhaust pipe L1 is provided with an exhaust valve V1.
  • the exhaust valve V1 is, for example, an electromagnetic valve.
  • the adsorption part R1 is connected to the vacuum pump 12, so that the culture vessel 11 is adsorbed on the support surface M1 side.
  • the exhaust pipe L2 is provided with a pressure gauge PG1.
  • the pressure gauge PG1 detects the negative pressure transmitted from the vacuum pump 12 to the adsorption unit R1.
  • the drive unit 3 is, for example, an electromagnetic vibration generator, and is installed on the installation table 10 via a vibration isolation member 31.
  • the anti-vibration member 31 is, for example, an anti-vibration rubber, and reduces the transmission of vibration from the drive unit 3 to the installation base 10.
  • the drive unit 3 includes a vibrating body 30 that protrudes upward. The vibrating body 30 supports the culture vessel holding table 2 through the connecting portion 9.
  • the connecting portion 9 has an adsorption portion R2 that adsorbs the bottom plate 7, and is fixed to the vibrating body 30.
  • the connecting portion 9 is connected to the vacuum pump 12 via the exhaust pipe L3.
  • the exhaust pipe L3 is provided with an exhaust valve V3 and a pressure gauge PG2.
  • the exhaust valve V3 is, for example, an electromagnetic valve. When the exhaust valve V3 is opened, the suction portion R2 is connected to the vacuum pump 12, so that the bottom plate 7 is sucked to the connecting portion 9.
  • the pressure gauge PG2 detects a negative pressure transmitted from the vacuum pump 12 to the adsorption unit R2.
  • the driving unit 3 vibrates the vibrating body 30 along the vertical direction.
  • the culture vessel holder 2 is also vibrated along the vertical direction.
  • the culture vessel 11 is vibrated so that the whole moves along the vertical direction to the same side simultaneously with the same amount of displacement.
  • the drive unit 3 vibrates the culture vessel holder 2 so that the entire culture vessel 11 moves to the same side at the same displacement amount in the direction orthogonal to the support surface M1.
  • the vibration direction of the culture vessel 11 is not necessarily along the vertical direction.
  • the culture vessel 11 may be displaced in the vertical direction and also in the horizontal direction. That is, the drive unit 3 may vibrate the vibrating body 30 along a direction inclined with respect to the vertical direction.
  • the displacement direction of each part of the culture container 11 needs to correspond, but the displacement amount may differ for every part.
  • the monitoring unit 4 is an imaging device such as a CCD camera, for example, and acquires an image of the shelf unit 6.
  • the control unit 5 controls the exhaust valves V1 and V3 and the drive unit 3 to execute the following processing.
  • the control unit 5 controls the exhaust valve V3 according to the user's instruction input, and the state where the suction part R2 of the connecting part 9 is connected to the vacuum pump 12 and the state where the suction part R2 is disconnected from the vacuum pump 12 Switch. Thereby, the drive part 3 and the culture container holding stand 2 are switched between a connected state and a separated state.
  • the control part 5 acquires the measurement value of the pressure gauge PG2. That is, the negative pressure transmitted from the vacuum pump 12 to the suction part R2 is acquired. If the measured value obtained from the pressure gauge PG2 is higher than the threshold value, it is determined that the adsorption of the culture vessel holder 2 is incomplete, and the user is notified by a monitor (not shown) or an alarm lamp (not shown), for example. To inform. That is, the control unit 5 functions as a connection abnormality detection unit.
  • the control unit 5 processes the image acquired by the monitoring unit 4 and detects whether or not the culture vessel 11 is placed on the shelf 6. And each exhaust valve V1 is controlled, the adsorption
  • the control unit 5 acquires the measurement value of the pressure gauge PG1. That is, the negative pressure transmitted from the vacuum pump 12 to the shelf 6 is acquired. If the measured value obtained from the pressure gauge PG1 is higher than the threshold value, it is determined that the adsorption of the culture vessel 11 is incomplete, and is notified to the user by, for example, a monitor (not shown) display or an alarm lamp (not shown). To do. That is, the control unit 5 functions as an adsorption abnormality detection unit.
  • the control unit 5 controls the drive unit 3 so as to vibrate the vibrating body 30 in a state where the culture vessel holding table 2 is adsorbed by the adsorption unit R2 and the culture vessel 11 is adsorbed by the adsorption unit R1.
  • the culture vessel holding table 2 vibrates, and accordingly, the culture vessel 11 on the shelf 6 also vibrates. Due to the vibration of the culture vessel 11, the cells in the culture vessel 11 are separated from the inner surface of the culture vessel 11.
  • the control unit 5 controls the drive unit 3 so as to keep the amplitude and frequency of the vibrating body 30 near the target value.
  • the frequency is 20 Hz to 2 kHz.
  • the frequency may be 20 to 100 Hz, 30 to 70 Hz, or 50 to 60 Hz.
  • the vibration isolating element for reducing vibration transmission tends to become more complex as the frequency decreases.
  • the power source of the drive unit becomes larger as the frequency becomes higher.
  • the frequency is 20 Hz to 2 kHz
  • the deposits on the inner surface of the culture vessel can be peeled off while suppressing the complexity of the vibration isolation element or the increase in the size of the power source of the drive unit.
  • the frequency is 20 to 100 Hz, the enlargement of the power source of the drive unit can be further suppressed.
  • the amplitude in the direction orthogonal to the support surface M1 is, for example, preferably greater than 0 mm and not greater than 5 mm, more preferably 1 to 4 mm, and particularly preferably 1 to 2 mm.
  • the amplitude is more than 0 mm and not more than 5 mm, the deposits on the inner surface of the culture vessel can be peeled off while suppressing an increase in size of the power source of the drive unit.
  • the amplitude means the total amplitude obtained by adding up the maximum displacement amount on one side and the maximum displacement amount on the other side with reference to the center position of vibration.
  • the vibration waveform of the vibrating body 30 is not particularly limited, but typical examples include a sine waveform, a rectangular waveform, a triangular waveform, and a sawtooth waveform. Although a sine wave is the most affordable from the viewpoint of ease of design of the drive unit, it is not limited to this.
  • the waveform means a waveform drawn on a graph with the horizontal axis representing elapsed time and the vertical axis representing displacement.
  • the shelf 6 has a rectangular shape in plan view, and is made of, for example, an aluminum-based metal material.
  • the spacers 20 are arranged at the four corners of the shelf 6.
  • an annular housing groove 6 d is formed on the support surface M ⁇ b> 1 of the shelf 6, and an annular seal ring 60 is housed in the housing groove 6 d.
  • the housing groove 6d and the seal ring 60 have a shape along the peripheral edge of the lower surface of the culture vessel 11.
  • the seal ring 60 is made of, for example, silicone rubber, and seals a gap between the lower surface of the culture vessel 11 and the support surface M1.
  • an inner region of the seal ring 60 is formed with five grooves 6a parallel to each other and five grooves 6b orthogonal to the grooves 6a.
  • an opening OP1 is formed on the support surface M1
  • a negative pressure space S1 is formed according to the depth of the grooves 6a and 6b.
  • the negative pressure space S1 is a space for applying a negative pressure, and communicates with the opening OP1.
  • the ten grooves 6a and 6b are connected to each other. Specifically, the outermost two grooves 6a intersect with the three grooves 6b, and the remaining three grooves 6a intersect with all the grooves 6b. The two outermost grooves 6b intersect with the three grooves 6a, and the remaining three grooves 6b intersect with all the grooves 6a. Thus, the groove 6a and the groove 6b are connected, the grooves 6a are connected via the groove 6b, and the grooves 6b are connected via the groove 6a.
  • Each groove 6a, 6b extends to the vicinity of the seal ring 60 within a range not reaching the accommodation groove 6d.
  • the opening OP ⁇ b> 1 is spread over a wide range in the region inside the seal ring 60. That is, the openings OP1 are distributed so as to match the shape and size of the culture vessel 11.
  • a vent hole 6c that opens the negative pressure space S1 downward is formed.
  • the ventilation hole 6c is located closer to the seal ring 60.
  • a connection portion 65 is provided in a portion corresponding to the ventilation hole 6 c, and a ventilation hole 65 a is formed in the connection portion 65.
  • the vent hole 65a opens upward and continues to the vent hole 6c.
  • the vent hole 65a opens to the outer peripheral side of the shelf 6, and the exhaust pipe L1 is connected to the outer peripheral side opening.
  • the adsorption part R1 described above is constituted by the seal ring 60, the opening OP1, the negative pressure space S1, and the vent holes 6c and 5a. That is, when the exhaust valve V1 of the exhaust pipe L1 is opened, the gas in the negative pressure space S1 is sucked out by the vacuum pump 12, and negative pressure is applied to the negative pressure space S1. When this negative pressure acts on the culture vessel 11 through the opening OP1, the culture vessel 11 is adsorbed on the support surface M1 side.
  • the vent holes 6c, 5a, the negative pressure space S1, and the opening OP1 function as a path for transmitting the negative pressure generated by the vacuum pump 12 to the support surface M1 side.
  • the seal ring 60 seals the gap between the lower surface of the culture vessel 11 and the support surface M1 around the opening OP1, and prevents the gas from flowing into the negative pressure space S1. For this reason, a negative pressure acts on the culture container 11 efficiently, and the culture container 11 is adsorb
  • a resin guide frame 62 for positioning the culture vessel 11 on the adsorption portion R1 is provided around the adsorption portion R1. That is, the culture vessel holder 2 further includes a guide frame 62.
  • the guide frame 62 has a U-shape that is open on one side of the shelf 6, and the culture vessel 11 can enter the guide frame 62 from the open end 62 a.
  • the culture vessel holder 2 further includes guide rails 63 and 64.
  • the guide rails 63 and 64 are located outside the suction portion R1 and are respectively along the guide frame 62.
  • the two guide rails 63 are positioned in the vicinity of the inner edge of the open end 62a.
  • the two guide rails 64 are located in the vicinity of the intermediate position between the open end portions 62a.
  • tapered portions 63a and 64a that are inclined so as to approach the support surface M1 toward the suction portion R1 side are formed on the suction portion R1 side.
  • the guide rails 63 and 64 are interposed between the culture vessel 11 moving from the open end 62a side onto the adsorption unit R1 and the support surface M1, and guide the culture vessel 11 onto the adsorption unit R1. Therefore, the moving culture vessel 11 and the support surface M1 are separated from each other by the guide rails 63 and 64. This prevents the culture container 11 from being caught by the seal ring 60 and the grooves 6a and 6b (components of the adsorption part R1), so that the culture container 11 can be smoothly moved from the open end 62a side onto the adsorption part R1. Can be transported.
  • the culture vessel 11 on the adsorption part R1 When the culture vessel 11 on the adsorption part R1 is moved to the open end 62a side, the culture vessel 11 is guided on the guide rails 63, 64 by the tapered surfaces 63a, 64a. For this reason, the culture container 11 on adsorption part R1 can be smoothly transferred to the open end part 62a side.
  • the bottom plate 7 has a rectangular shape corresponding to the shelf 6, and the spacers 21 are arranged at the four corners of the shelf 6 and the bottom plate 7.
  • the bottom plate 7 is made of, for example, an aluminum metal material.
  • the bottom plate 7 is provided with two positioning holes 7a. The two positioning holes 7 a are aligned in the diagonal direction of the bottom plate 7 and are respectively positioned in the vicinity of the spacer 21.
  • the connecting portion 9 has a rectangular shape corresponding to the bottom plate 7 and supports the bottom plate 7. That is, the connection part 9 has the support surface M2 which supports the culture container holding stand 2.
  • the connecting portion 9 is made of, for example, an aluminum-based metal material.
  • a positioning protrusion 93 is provided at a position corresponding to the positioning hole 7a in the support surface M2. The bottom plate 7 and the connecting portion 9 are positioned with respect to each other by fitting the positioning holes 7 a and the positioning protrusions 93.
  • An annular housing groove 9e is formed in a region of the support surface M2 between the positioning protrusions 93, and an annular seal ring 90 is housed in the housing groove 9e.
  • the seal ring 90 is made of, for example, silicone rubber, and seals a gap between the lower surface of the bottom plate 7 and the support surface M2.
  • a circular recess 9a is formed in the inner surface of the seal ring 90 in the support surface M2.
  • an opening OP2 is formed in the support surface M2, and a negative pressure space S2 is formed according to the depth of the recess 9a.
  • the negative pressure space S2 is a space for applying a negative pressure, and communicates with the opening OP2.
  • a plurality of bolt holes 9b are formed in the center of the bottom of the recess 9a, and a counterbore hole 9c is formed in the upper part of the bolt holes 9b.
  • Bolts 91 are passed through the bolt holes 9b from above.
  • the tip of the bolt 91 passes through the bolt hole 9b and is screwed into the vibrating body 30, and the head of the bolt 91 is received in the counterbore hole 9c.
  • the connecting portion 9 is fixed to the vibrating body 30 by tightening the bolt 91 screwed into the vibrating body 30.
  • the gaps between the bolt holes 9b and counterbore holes 9c and the bolts 91 are filled with a sealant.
  • a vent 9d that opens the negative pressure space S2 downward is formed in a region outside the vibrating body 30 in the bottom of the recess 9a.
  • a connection portion 92 is provided in a portion corresponding to the air hole 9 d, and the air hole 92 a is formed in the connection portion 92.
  • the ventilation hole 92a opens upward and continues to the ventilation hole 9d.
  • the ventilation hole 92a opens to the outer peripheral side of the connecting portion 9, and the exhaust pipe L3 is connected to the opening on the outer peripheral side.
  • the seal ring 90, the opening OP2, the negative pressure space S2, and the vent holes 9d and 92a constitute an adsorption portion R2 that adsorbs the bottom plate 7. That is, when the exhaust valve V3 of the exhaust pipe L3 is opened, the gas in the negative pressure space S2 is sucked out by the vacuum pump 12, and negative pressure is applied to the negative pressure space S2. When this negative pressure acts on the bottom plate 7 through the opening OP2, the bottom plate 7 is adsorbed to the support surface M2 side.
  • the vent holes 9d and 92a, the negative pressure space S2, and the opening OP2 function as a path for transmitting the negative pressure generated by the vacuum pump 12 to the support surface M1 side.
  • the seal ring 90 seals a gap between the lower surface of the bottom plate 7 and the support surface M2 around the opening OP2, and prevents gas from flowing into the negative pressure space S2. For this reason, a negative pressure acts on the baseplate 7 efficiently, and the baseplate 7 is adsorb
  • the bolt hole 9b is provided at the bottom of the recess 9a. That is, the fixed portion between the connecting portion 9 and the vibrating body 30 is located in the region of the negative pressure space S2.
  • the sealing agent filled in the gaps between the bolt holes 9b and the counterbore holes 9c and the bolts 91 seals the gaps generated to form the fixing portion. This also contributes to securing the suction force of the bottom plate 7.
  • the culture vessel 11 can be vibrated with a simple configuration in which the culture vessel holding base 2 is vibrated by the drive unit 3.
  • the culture vessel 11 is vibrated so that the whole moves simultaneously with the same amount of displacement to the same side in the direction orthogonal to the support surface M1. Due to this vibration, the deposit on the inner surface of the culture vessel 11 is efficiently peeled off. Therefore, the operation of peeling the deposits on the inner surface of the culture vessel 11 can be automated with a simple configuration.
  • the culture vessel holding stand 2 has a plurality of shelves 6 each constituting a support surface M1. For this reason, it is possible to arrange a plurality of culture vessels 11 so as to overlap each other along the direction orthogonal to the support surface M1, and to vibrate these culture vessels 11 simultaneously. Thereby, the deposit
  • the culture vessel holder 2 has a larger support surface M1 than the lower surface of the culture vessel 11. For this reason, it is easy to move the entire culture vessel 11 to the same side at the same time with the same amount of displacement.
  • the drive unit 3 vibrates the culture vessel holder 2 along a direction orthogonal to the support surface M1. That is, since the driving unit 3 vibrates the culture vessel holding table 2 along the direction in which the shelf 6 overlaps, the inertial force hardly acts in the horizontal direction perpendicular to the direction in which the shelf 6 overlaps, and the culture vessel holding table 2 Is hard to bend. For this reason, the vibration state for every culture container 11 is hard to vary, and the variation in peelability for every culture container 11 is reduced.
  • the adsorption part R1 of the culture vessel holder 2 adsorbs the culture vessel 11 to the support surface M1 side by negative pressure.
  • suction part R1 can be comprised only by providing the path
  • the culture vessel 11 can be reliably held by adsorption. Therefore, the culture vessel 11 being driven can be reliably held with a simple configuration.
  • the path for transmitting negative pressure to the support surface M1 includes the opening OP1 and the negative pressure space S1, and the opening OP1 and the negative pressure space S1 are configured by grooves 6a and 6b. Since both the opening OP1 and the negative pressure space S1 can be configured by the grooves 6a and 6b, the configuration of the suction portion R1 can be further simplified. Further, by forming the plurality of grooves 6a and 6b connected to each other, the distribution of the openings OP1 can be freely adjusted according to the shape and size of the culture vessel 11, and the culture vessel 11 can be held more reliably.
  • the culture vessel drive device 1 further includes an adsorption abnormality detection unit (control unit 5) that detects incomplete adsorption of the culture vessel 11 based on the detection result by the pressure gauge PG1. For this reason, incomplete adsorption
  • control unit 5 adsorption abnormality detection unit
  • the adsorption part R1 is provided for each shelf part 6. For this reason, the several culture container 11 can be reliably hold
  • the culture vessel drive device 1 further includes a connecting portion 9.
  • the connection unit 9 is fixed to the drive unit 3 and connects the culture vessel holding table 2 and the drive unit 3 by adsorbing the culture vessel holding table 2. For this reason, while the culture vessel 11 is held, the culture vessel holding stand 2 can be separated from the drive unit and more various processes can be performed. For example, as shown in FIG. 6, the culture vessel holding table 2 as a whole can be tilted to perform a process of discharging the culture solution and cells from the liquid feeding port 11 a of each culture vessel 11. Moreover, the structure of the culture vessel drive device 1 can be further simplified by using one vacuum pump 12 as both the adsorption part R1 of the shelf 6 and the adsorption part R2 of the connecting part 9.
  • the connecting part 9 may be fixed to the culture vessel holding table 2 and adsorb the driving part 3.
  • the culture vessel holder 2A shown in FIGS. 7 and 8 is obtained by replacing the adsorption part R1 constituted by the grooves 6a and 6b with an adsorption part R3 constituted by a plurality of holes 6e and a cavity 6f.
  • the cavity 6f is provided in the shelf 6A of the culture vessel holder 2A and extends over the entire region surrounded by the seal ring 60.
  • Each hole 6e is formed above the shelf 6A in a region surrounded by the seal ring 60, and opens to the support surface M1 and the cavity 6f. That is, the plurality of holes 6e are formed in the support surface M1 and communicated with each other through the cavity 6f.
  • a plurality of openings OP3 are formed in the support surface M1, and one negative pressure space S3 is formed by the plurality of holes 6e and the cavity 6f.
  • the negative pressure space S3 is a space for applying a negative pressure, and communicates with the opening OP3.
  • the holes 6e are arranged so as to be scattered from the vicinity of the center of the region surrounded by the seal ring 60 to the vicinity of the peripheral edge and to the entire periphery of the seal ring 60. Thereby, in the area
  • a vent 6g that opens the negative pressure space S3 downward is formed at the bottom of the shelf 6A.
  • the vent hole 6g is located near the periphery of the cavity 6f and is continuous with the vent hole 65a of the connecting portion 65.
  • the opening OP3 and the negative pressure space S3 function in the same manner as the opening OP1 and the negative pressure space S1. That is, in the culture vessel holder 2A, the adsorbing portion R3 that adsorbs the culture vessel 11 includes the seal ring 60, the opening OP3, the negative pressure space S3, and the vent holes 6g and 5a.
  • the culture vessel holding table 2A can reliably hold the driving culture vessel 11 with a simple configuration. Further, by adjusting the number and arrangement of the holes 6e, the distribution of the openings OP3 can be freely adjusted according to the shape and size of the culture vessel 11, and the culture vessel 11 can be held more reliably.
  • the holding part of the shelf part 6 may hold the culture vessel 11 by means other than adsorption, such as a gripping mechanism or a locking mechanism.
  • the suction part of the connecting part 9 may be constituted by a plurality of grooves similarly to the suction part R1 of the shelf part 6, or may be constituted by a plurality of holes and cavities similarly to the suction part R3 of the shelf part 6A.
  • the connection part 9 may hold
  • the culture system 100 includes a culture vessel drive device 1, a culture vessel 11, an infusion device 200, a scraper 13, a scraper drive device 300, an incubator 400, and a system control unit 110. .
  • the infusion device 200 includes supply containers 201A and 201B, a recovery container 202, and liquid feeding pipes L4, L5, L6, L7, and L8, and supplies liquid to the culture container 11 or liquid from the culture container 11 Is discharged.
  • the supply container 201 ⁇ / b> A contains a culture solution for supply to the culture container 11.
  • the culture solution is a liquid containing components that serve as nutrients for the culture target.
  • the supply container 201 ⁇ / b> B stores a cleaning liquid for supply to the culture container 11.
  • the washing solution is, for example, a pH buffer solution.
  • the collection container 202 stores the liquid discharged from the culture container 11.
  • the liquid feeding tube L4 is connected to the liquid feeding port 11a of the culture vessel 11.
  • the liquid supply pipe L5 is branched into three liquid supply pipes L6, L7, and L8, the liquid supply pipe L6 is connected to the supply container 201A, the liquid supply pipe L7 is connected to the supply container 201B, and the liquid supply pipe L8 is recovered.
  • container 202 Connected to container 202. That is, the liquid supply pipes L4, L5, L6, L7, and L8 connect the culture container 11, the supply containers 201A and 201B, and the recovery container 202.
  • the liquid supply pipes L4, L6, L7, and L8 are provided with liquid supply valves V4, V6, V7, and V8, respectively.
  • the liquid feeding valves V4, V6, V7, V8 are, for example, electromagnetic valves.
  • the liquid supply pipe L5 is provided with a pump P1. When supplying the liquid to the culture container 11, the pump P1 pumps the liquid from the supply containers 201A and 201B to the culture container 11 side. When discharging the liquid from the culture vessel 11, the pump P1 pumps the liquid from the culture vessel 11 side to the collection vessel 202 side.
  • the scraper 13 is enclosed in the culture vessel 11 and attached to the center of the bottom of the culture vessel 11.
  • the scraper 13 is rotatable about an axis orthogonal to the bottom surface of the culture vessel 11, and has a pair of rotating arms 13c extending from the rotation axis to opposite sides.
  • a pair of permanent magnets 13a is provided on the top of the pair of rotating arms 13c.
  • a pair of blades 13b are respectively provided below the pair of rotating arms 13c. The blade 13 b projects downward from the rotary arm 13 c and contacts the bottom surface of the culture vessel 11.
  • the scraper driving device 300 is installed around the culture vessel driving device 1 and has four support arms 301 extending to the culture vessel driving device 1 side.
  • the four support arms 301 are lined up and down so as to correspond to the four stages of the shelf sections 6 respectively.
  • a drive rotator 302 is attached on the tip of each support arm 301.
  • the drive rotator 302 is rotatable about an axis extending in the vertical direction, and has a pair of rotating arms 302b extending from the rotation axis to opposite sides.
  • a pair of permanent magnets 302a are respectively provided on the top of the pair of rotating arms 302b.
  • the scraper driving device 300 incorporates an actuator for extending and retracting the support arm 301, and by extending and retracting the support arm 301, a drive position (see FIG. 11) below the shelf 6 and a standby position around the shelf 6.
  • the drive rotator 302 is transferred between them.
  • the scraper driving device 300 has a built-in actuator that rotates the driving rotating body 302 disposed under the shelf 6.
  • the permanent magnet 302a When the drive rotor 302 is in the drive position, the permanent magnet 302a generates a magnetic force in cooperation with the permanent magnet 13a. Power is transmitted from the drive rotor 302 to the scraper 13 by this magnetic force. For this reason, when the drive rotator 302 rotates, the scraper 13 rotates accordingly. That is, the scraper driving device 300 drives the scraper 13 from the outside of the culture vessel 11 by magnetic force.
  • the shelf 6 is preferably made of a nonmagnetic material such as an aluminum-based metal so that the magnetic force between the permanent magnet 13a and the permanent magnet 302a is not weakened.
  • the permanent magnet does not necessarily have to be disposed on both the scraper 13 and the drive rotor 302, and a soft magnetic material such as iron is disposed on one of the scraper 13 and the drive rotor 302 instead of the permanent magnet. May be.
  • the incubator 400 is a thermostatic device having a plurality of cells C1 arranged in the vertical direction, and is provided independently of the culture vessel driving device 1. Each cell C1 accommodates the culture vessel 11.
  • the incubator 400 promotes culture in the culture vessel 11 by keeping the inside of the cell C1 at a temperature suitable for culture.
  • the system control unit 110 is configured by a computer, for example, and automatically executes a part of the culture procedure by controlling the culture vessel driving device 1, the infusion device 200, and the scraper driving device 300.
  • the user installs the culture vessel 11 on the four-stage shelf 6 of the culture vessel drive device 1 (S01), and feeds the liquid feed pipe L4 to the liquid feed port 11a of each culture vessel 11.
  • S01 the culture vessel drive device 1
  • S02 the liquid feed pipe L4 to the liquid feed port 11a of each culture vessel 11.
  • a plurality of types of cells including a culture target and a scraper 13 are enclosed in advance.
  • monocytes and lymphocytes are encapsulated as an example of a plurality of types of cells.
  • the system control unit 110 controls the culture vessel driving device 1 so as to hold the culture vessel 11.
  • the system control unit 110 controls the infusion device 200 so as to supply the cleaning solution to the culture vessel 11 without opening the culture vessel 11 (S03). Specifically, by controlling the infusion device 200, the liquid feeding valves V4 and V7 are opened and the liquid feeding valves V6 and V8 are closed, and the cleaning liquid is pumped from the supply container 201B to the culture container 11 by the pump P1. .
  • the system control unit 110 executes first vibration control for controlling the drive unit 3 so as to vibrate the vibrating body 30 (S04).
  • the frequency and amplitude of vibration are set so that more lymphocytes can be detached from the inner surface of the culture vessel 11 than monocytes. That is, the system control unit 110 functions as a vibration peeling control unit and vibrates the culture container holder 2 so as to vibrate the culture container holding base 2 under the condition that the deposits on the inner surface of the culture container 11 can be peeled without opening the culture container 11.
  • the drive device 1 is controlled.
  • the system control unit 110 as a vibration detachment control unit controls the culture container driving device 1 so as to vibrate the culture container holding table 2 under the condition that more lymphocytes than monocytes can be detached from the inner surface of the culture container 11. To do.
  • the system control unit 110 controls the infusion device 200 so as to discharge the suspension containing the detached lymphocytes from the culture vessel 11 (S05). Specifically, the infusion device 200 is controlled so that the suspension is pumped from the culture vessel 11 to the collection vessel 202 by the pump P1 with the liquid feeding valves V4 and V8 opened and the liquid feeding valves V6 and V7 closed. To do. The suspension recovered in the recovery container 202 is discarded.
  • the culture vessel holder 2 may be separated from the drive unit 3 in discharging the washing solution, and the culture vessel holding table 2 and the drive unit 3 may be connected again after the washing solution is discharged. Thereby, when discharging
  • the process of tilting the culture vessel holder 2 may be performed manually by the user or automatically by a dedicated device.
  • the system control unit 110 controls the infusion device 200 so as to supply the culture solution to the culture vessel 11 without opening the culture vessel 11 (S06). Specifically, the infusion device 200 is controlled so that the culture solution is pumped from the supply container 201B to the culture vessel 11 by the pump P1 with the solution delivery valves V4 and V6 opened and the solution delivery valves V7 and V8 closed. To do.
  • the system control unit 110 controls the culture vessel holder 2 so as to release the hold of the culture vessel 11.
  • the user removes the liquid feeding tube L4 from the liquid feeding port 11a of the culture container 11, transfers the culture container 11 to the incubator 400 (S07), and accommodates it in the cell C1 of the incubator 400.
  • the temperature in the cell C1 is maintained at a temperature suitable for monocyte culture by the incubator 400, and monocyte culture is promoted. Thereby, dendritic cells derived from monocytes (monocyte differentiation) are cultured (S08).
  • the user transfers the culture vessel 11 after completion of the culture to the culture vessel drive device 1 and re-installs the culture vessel 11 after completion of the culture on the four-stage shelf 6 (S09).
  • the liquid supply pipe L4 is connected to the liquid supply port 11a (S10).
  • the system control unit 110 controls the culture vessel driving device 1 so as to hold the culture vessel 11.
  • the system control unit 110 executes second vibration control for controlling the drive unit 3 so as to vibrate the vibrating body 30 (S11).
  • the frequency and amplitude of the vibration are set so that the dendritic cells can be detached from the inner surface of the culture vessel 11. That is, the system control unit 110 serving as the vibration detachment control unit vibrates the culture container holding table 2 under the condition that the differentiated monocytes can be separated from the inner surface of the culture container 11 after culturing in the incubator 400.
  • the drive device 1 is further controlled.
  • the system control unit 110 controls the infusion device 200 so as to discharge the suspension containing the detached dendritic cells from the culture vessel 11 (S12). Specifically, the infusion device 200 is controlled so that the suspension is pumped from the culture vessel 11 to the collection vessel 202 by the pump P1 with the liquid feeding valves V4 and V8 opened and the liquid feeding valves V6 and V7 closed. To do. As a result, the dendritic cells detached from the inner surface of the culture vessel 11 are collected in the collection vessel 202.
  • the culture vessel holder 2 may be separated from the drive unit 3 in discharging the suspension, and the culture vessel holder 2 and the drive unit 3 may be connected again after the suspension is discharged.
  • the entire culture vessel holder 2 can be tilted so that the liquid feeding port 11a of each culture vessel 11 is on the lower side (see FIG. 6).
  • the process of tilting the culture vessel holder 2 may be performed manually by the user or automatically by a dedicated device.
  • the system control unit 110 controls the infusion device 200 so as to supply the cleaning solution to the culture vessel 11 without opening the culture vessel 11 (S13).
  • the infusion apparatus 200 is controlled so that the cleaning liquid is pumped from the supply container 201B to the culture container 11 by the pump P1 with the liquid feeding valves V4 and V7 opened and the liquid feeding valves V6 and V8 closed. .
  • the system control unit 110 controls the scraper driving device 300 so as to transfer and rotate the driving rotating body 302 to the driving position without opening the culture vessel 11 (S14, see FIG. 11).
  • the scraper 13 is rotationally driven, and the blade 13 b of the scraper 13 scratches the inner surface of the culture vessel 11. That is, the system control unit 110 functions as a scraping control unit that controls the scraper driving device 300 so that the scraper 13 scrapes off the deposits on the inner surface of the culture vessel 11.
  • the system control unit 110 controls the infusion device 200 so as to discharge the suspension containing the dendritic cells scraped off from the culture vessel 11 (S15).
  • the infusion device 200 is controlled so that the suspension is pumped from the culture vessel 11 to the collection vessel 202 by the pump P1 with the liquid feeding valves V4 and V8 opened and the liquid feeding valves V6 and V7 closed. To do. Thereby, the dendritic cells scraped off from the inner surface of the culture vessel 11 are collected in the collection vessel 202.
  • the system controller 110 as the vibration separation controller controls the culture container drive device 1 to peel off the deposits on the inner surface of the culture container 11. Can be collected efficiently. Further, by controlling the scraper driving device 300 by the system control unit 110 as a scraping control unit, the deposits remaining on the inner surface of the culture vessel 11 can be scraped off and collected. Therefore, more deposits can be collected efficiently.
  • the system controller 110 as a vibration peeling controller is configured to vibrate the culture vessel holder 2 under the condition that the deposits on the inner surface of the culture vessel 11 can be peeled off without opening the culture vessel 11. To control. For this reason, the deposit
  • the culture system 100 further includes an infusion device 200 that supplies a liquid to the culture container 11 or discharges the liquid from the culture container 11 without opening the culture container 11 held on the culture container holder 2. . Therefore, without opening the culture vessel 11, a step of supplying a liquid such as a culture solution into the culture vessel 11, a step of peeling off the deposits on the inner surface of the culture vessel 11, and collecting the peeled deposits together with the liquid Can be performed. For this reason, culture
  • the scraper 13 is accommodated in the culture vessel 11, and the scraper driving device 300 drives the scraper 13 from the outside of the culture vessel 11 by magnetic force. For this reason, the process of scraping off and collecting the deposits on the inner surface of the culture vessel 11 can be further performed without opening the culture vessel 11. For this reason, collection
  • the system control unit 110 as a vibration detachment control unit vibrates the culture container holding table 2 under conditions that allow more lymphocytes than monocytes to be detached from the inner surface of the culture container 11 before culturing in the incubator 400.
  • the culture container driving device 1 is controlled. By exfoliating and removing lymphocytes adhering to the inner surface of the culture vessel 11, the monocyte culture in the incubator 400 can be promoted after sufficiently increasing the ratio of monocytes in the culture vessel 11. Thereby, monocyte-derived dendritic cells can be cultured efficiently.
  • the system control unit 110 as a vibration detachment control unit controls the culture container driving device 1 so that the culture container holding table 2 is vibrated under conditions that allow dendritic cells to be detached from the inner surface of the culture container 11 after culturing in the incubator 400. Further control. Thereby, in addition to being able to culture
  • the incubator 400 and the culture vessel driving device 1 are provided independently of each other. For this reason, the culture
  • the culture vessel holding table 2 does not necessarily have a plurality of shelves.
  • Example 1 For cell A, four culture containers 11 in which a certain amount of cells are seeded and adhered only to the center (spot a) of the bottom of the culture container, and only a certain amount of cells are on the outermost end (spot b) of the bottom of the culture container. Four culture vessels 11 seeded and adhered to each other and four culture vessels 11 to which a predetermined amount of cells were seeded and adhered only between the spots a and b (spot c) were prepared.
  • cell B four culture containers 11 in which a certain amount of cells are seeded and adhered only to spot a, four culture containers 11 in which a certain amount of cells are seeded and adhered only to spot b, a certain amount of Four culture vessels 11 in which cells were seeded and adhered only to spot c were prepared.
  • Cell A is a lymphocyte and cell B is a Ball-1 cell.
  • Each culture vessel 11 was placed on the shelf 6 of the culture vessel holder 2 and a cleaning solution was supplied into each culture vessel 11. Then, the culture vessel holder 2 was vibrated so that the culture vessel 11 vibrates along the vertical direction as shown in FIG.
  • the vibration waveform was a sine waveform, the frequency was about 53 Hz, and the amplitude was about 1.7 mm. After stopping the vibration of the culture vessel holder 2, the suspension containing the cells was collected from each culture vessel 11.
  • Example 2 In the same manner as in Example 1, 24 culture vessels 11 were prepared. Suspensions containing cells were collected from within each culture vessel 11 under the same conditions as in Example 1 except that the vibration frequency of the culture vessel holder 2 was about 20 Hz and the amplitude was about 5 mm.
  • Example 3 In the same manner as in Example 1, 24 culture vessels 11 were prepared. Suspensions containing cells were collected from within each culture vessel 11 under the same conditions as in Example 1 except that the frequency of vibration of the culture vessel holder 2 was about 100 Hz and the amplitude was about 0.6 mm.
  • Example 4 In the same manner as in Example 1, 24 culture vessels 11 were prepared. Suspensions containing cells were collected from within each culture vessel 11 under the same conditions as in Example 1 except that the frequency of vibration of the culture vessel holder 2 was about 2 kHz and the amplitude was about 0.05 mm.
  • Example 1 In the same manner as in Example 1, 24 culture vessels 11 were prepared. As shown in FIG. 12 (b), the culture vessel holder 2 is vibrated so that the culture vessel 11 vibrates along a horizontal straight line, the vibration frequency is about 3.3 Hz, and the amplitude is about 20 mm. In the same conditions as in Example 1, a suspension containing cells was recovered from each culture vessel 11.
  • Example 2 In the same manner as in Example 1, 24 culture vessels 11 were prepared. As shown in FIG. 12 (c), the culture vessel holder 2 is vibrated so that the central portion of the culture vessel 11 periodically draws a circle, the vibration frequency is about 33 Hz, and the central portion of the culture vessel 11 is drawn. A suspension containing cells was collected from each culture vessel 11 under the same conditions as in Example 1 except that the radius of the circle was about 2 mm.
  • Example 3 In the same manner as in Example 1, 24 culture vessels 11 were prepared. As shown in FIG. 12 (d), the culture vessel holder 2 is vibrated so that the culture vessel 11 periodically tilts about an axis that horizontally crosses the culture vessel 11, and the vibration frequency is about 2 Hz. A suspension containing cells was collected from each culture vessel 11 under the same conditions as in Example 1 except that the amplitude of the tilt angle was about 10 °.
  • Example 4 In the same manner as in Example 1, 24 culture vessels 11 were prepared, and a washing solution was supplied into each culture vessel 11. After a vertical impact force was partially repeatedly applied to the bottom surface of each culture vessel 11 by the culture vessel drive device 500 shown in FIG. 13, the suspension containing the cells was collected from within each culture vessel 11. As shown in FIG. 13, the culture vessel drive device 500 includes a support plate 510 that supports the culture vessel 11 and a main body 520 that is disposed below the support plate 510.
  • the main body 520 has plungers 521 and 522 that are aligned in the horizontal direction and protrude upward, and the plungers 521 and 522 are alternately pushed up to collide with the support plate 510, thereby repeatedly applying an impact force to the lower surface of the culture vessel 11.
  • the frequency of repeated impact forces was about 20 Hz.
  • the strength of the impact force was adjusted so that the width of the vertical movement of the culture vessel 11 was 5 mm.
  • Example 5 In the same manner as in Example 1, 24 culture vessels 11 were prepared, and a washing solution was supplied into each culture vessel 11. After applying ultrasonic vibration to each culture vessel 11, a suspension containing cells was collected from each culture vessel 11. The frequency of the ultrasonic wave was about 20 kHz, and the amplitude was 0.01 mm.
  • Example 6 (Comparative Example 6) In the same manner as in Example 1, 24 culture vessels 11 were prepared, and a washing solution was supplied into each culture vessel 11. Each culture vessel 11 was manually pipetted to collect a suspension containing cells from the culture vessel 11. The linear velocity of the liquid supplied by pipetting (flow velocity along the inner surface of the culture vessel 11) was about 20 cm / sec.
  • the average value a of the residual amount at spot a, the average value b of the residual amount at spot b, and the average value c of the residual amount at spot c were determined. . Then, a ratio of the maximum value of the average values a to c to the minimum value of the average values a to c is calculated (hereinafter, this calculated value is referred to as “uniformity evaluation value”), and the peeling is performed depending on the size of the uniformity evaluation value. Was evaluated for uniformity.
  • the peelability evaluation results are shown in FIG. “OK” in FIG. 14 means that the peelability evaluation value is less than 1.2, and “NG” means that the peelability evaluation value is 1.2 or more.
  • the peelability evaluation values of Comparative Examples 1 to 3 were all 1.3 or more, whereas the peelability evaluation values of Examples 1 to 4 were all less than 1.1. .
  • the reason for this is estimated as follows. In Examples 1 to 4, the entire culture vessel 11 moves simultaneously on the same side with the same amount of displacement in the direction orthogonal to the support surface M1. When such vibration occurs, a Faraday wave WF is generated on the liquid surface in the culture vessel 11 as shown in FIG.
  • the evaluation results of the uniformity of peeling are shown in FIG. “OK” in FIG. 15 means that the uniformity evaluation value is less than 1.2, and “NG” means that the uniformity evaluation value is 1.2 or more.
  • the uniformity evaluation values of Comparative Examples 1 to 4 were all 1.3 or more, whereas the uniformity evaluation values of Examples 1 to 4 were all less than 1.1. .
  • the reason for this is estimated as follows. That is, as in Comparative Examples 1 to 3, in the system in which the culture vessel 11 is vibrated along the horizontal direction, the movement of the liquid in the culture vessel 11 is likely to be non-uniform, and cell detachment is likely to be non-uniform.
  • FIG. 16 shows the evaluation results of cell viability.
  • the cell viability in Examples 1 to 4 and Comparative Examples 1 to 4 are all close to 100%, whereas the cell viability in Comparative Example 5 is remarkably low. It was.
  • the reason for this is estimated as follows. That is, in Comparative Example 5, some cells were crushed by the energy of ultrasonic vibration. In contrast, in Examples 1 to 4 and Comparative Examples 1 to 4, the frequency at which the culture vessel vibrates is much smaller than the frequency of the ultrasonic wave, so that the energy applied to the cells is greater than when the culture vessel is vibrated ultrasonically. Smaller cells were crushed by vibration.
  • Example 1 [Evaluation of influence on frequency and amplitude]
  • the frequency and amplitude were changed to various values, and the cell detachability was compared.
  • 18 and 19 are diagrams in which circles having a diameter proportional to the peelability evaluation value are arranged on frequency and amplitude coordinates.
  • the horizontal axis and the vertical axis are logarithmic.
  • a curve indicated by a one-dot chain line in FIGS. 18 and 19 is an approximate curve connecting circles having a peelability evaluation value of about 1.
  • a curve indicated by a solid line is an approximate curve connecting circles corresponding to a peelability evaluation value of about 0.7.
  • a curve indicated by a broken line is an approximate curve connecting circles corresponding to a peelability evaluation value of about 1.2.
  • the frequency and amplitude are set so as to be on the upper right side of the curve indicated by the alternate long and short dash line, it is estimated that the same or better peelability can be obtained for pipetting. From this, according to the vibration states of Examples 1 to 4, it is possible to select a wide range of frequencies and amplitudes for obtaining a peelability equivalent to or better than pipetting, and depending on the characteristics of the device, the frequency and amplitude It was confirmed that can be set flexibly.
  • the present disclosure can be used for a system for culturing cells.
  • SYMBOLS 1 Culture container drive device, 2, 2A ... Culture container holding stand, 3 ... Drive part, 6, 6A ... Shelf part, 11 ... Culture container, 13 ... Scraper, 100 ... Culture system, 110 ... System control part (vibration peeling) Control unit, scraping control unit), 200 ... infusion device, 300 ... scraper driving device, 400 ... incubator, M1 ... support surface, R1 ... adsorption unit (holding unit).

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Abstract

Le dispositif d'entraînement de récipients de culture (1) est équipé : d'un support de récipients de culture (2) qui possède une face maintien (M1) maintenant des récipients de culture (11), et une partie adsorption (R1) supportant les récipients de culture (11) sur la face maintien (M1) ; et d'une partie entraînement (3) qui fait vibrer, dans une direction perpendiculaire à la face maintien (M1), le support de récipients de culture (2) selon une fréquence de 20Hz à 2kHz de sorte que l'ensemble des récipients de culture (11) bouge d'une même quantité de déplacement, en même temps et du même côté.
PCT/JP2014/062353 2013-09-06 2014-05-08 Dispositif d'entraînement de récipients de culture, et système de culture WO2015033616A1 (fr)

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JP2018102298A (ja) * 2016-12-27 2018-07-05 学校法人慶應義塾 細胞処理装置及び細胞処理方法
KR20200050022A (ko) * 2018-10-31 2020-05-11 한국생산기술연구원 진공 세포 배양장치
JP2021035659A (ja) * 2019-08-30 2021-03-04 株式会社アクトラス 攪拌方法及び攪拌装置
CN113847942A (zh) * 2020-06-26 2021-12-28 株式会社日立制作所 数字化系统
WO2024079759A3 (fr) * 2022-10-13 2024-07-04 Omnibrx Biotechnologies Private Limited Bioréacteur de culture cellulaire avec dispositif de collecte de cellules et procédé de collecte de cellules

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JP2018102298A (ja) * 2016-12-27 2018-07-05 学校法人慶應義塾 細胞処理装置及び細胞処理方法
JP7083439B2 (ja) 2016-12-27 2022-06-13 慶應義塾 細胞処理装置及び細胞処理方法
KR20200050022A (ko) * 2018-10-31 2020-05-11 한국생산기술연구원 진공 세포 배양장치
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JP2021035659A (ja) * 2019-08-30 2021-03-04 株式会社アクトラス 攪拌方法及び攪拌装置
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CN113847942A (zh) * 2020-06-26 2021-12-28 株式会社日立制作所 数字化系统
WO2024079759A3 (fr) * 2022-10-13 2024-07-04 Omnibrx Biotechnologies Private Limited Bioréacteur de culture cellulaire avec dispositif de collecte de cellules et procédé de collecte de cellules

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